1. Field of the Invention
The present invention relates to the design, synthesis, and use of peptide modulators of ion channels. The peptides of the instant invention are useful in modulating ion-trafficking across potassium channels, specifically calcium activated potassium channels, such as BK and SK channels. More specifically, the present invention is drawn to the use of such peptide modulators in the treatments of pathophysiological conditions that are caused or characterized by electrolyte imbalances associated with calcium activated potassium channels.
2. Description of the Related Art
Large-conductance Ca2+-activated K+ channels (BKCa) channels whose α-subunits are encoded by the vertebrate Slo1 gene (which is also known as KCNMA1 and KCa1.1) are expressed in a wide range of tissues, including neurons and neuroendocrine cells, smooth muscle cells, endothelial and epithelial cells, and even in bone. The function of these channels is to cause cell hyperpolarization in response to physiological stimuli that elevate intracellular free Ca2+ and that depolarize cells. BKCa channels are often part of a negative feedback system that terminates Ca2+ influx. As such, they can produce protective effects on cells that are damaged by prolonged elevations of intracellular Ca2+. These channels also tend to cause relaxation of smooth muscles and general inhibition of the activity of other cell types by preventing their depolarization and by opposing Ca2+ influx.
Functional BKCa channels contain four pore-forming α-subunits. Each BKCa α-subunit (Slo1 protein) has seven membrane-spanning α-helices with an extracellular NH2 terminal domain, and a large globular cytopasmic COOH-terminal domain. BKCa channels become active when Ca2+ ions bind to specific sites on the cytoplasmic COOH-terminal domains of Slo1. Single-molecule measurements indicate that the opening of one BKCa channel causes a membrane conductance of 180-200 pS when membranes are in symmetrical 150 mM KCl (a standard assay condition, but one that is not physiological).
The broad importance of BKCa channels is highlighted by the phenotypes of knockout mouse models, which are mainly knockouts of the Slo1 gene and which have numerous defects including abnormal blood pressure control, altered endocrine status, deafness, alterations in smooth muscle and renal function, including bladder dysfunction, erectile dysfunction and abnormal electrolyte secretion by renal tubules, and several neurological problems. A Slo1 mutation in humans causes a coexistent generalized epilepsy and paroxysmal dyskinesia. On the basis of those observations, there is reason to believe that agents that increase the function of BKCa channels could be therapeutically useful in conditions including but not limited to epilepsy, chronic pain, migraine, asthma, chronic obstructive pulmonary disease, urinary incontinence, hypertension, erectile dysfunction, irritable bowel syndrome, renal disorders of electrolyte imbalance, and possibly in certain kinds of cancer.
Proper trafficking of BKCa channels is crucial and is a key regulatory step controlling their functional expression on the cell surface. This process is dependent on interactions of various protein binding partners with Slo1 variants inside cells, which regulate trafficking to the cell surface. In some tissues, for example brain and kidney BKCa α-subunits (Slo1 proteins) occur in two general classes that differ at the extreme COOH-terminal. These include short forms that have been called Slo1QEERL (where QEERL=glutamine-glutamate-glutamate-argine-lysine) and long forms referred to as Slo1VEDEC. The subscripts refer to the last five amino acid residues in each class of variants. Slo1VEDEC has a tail that extends 56 residues past the point where it diverges from Slo1QEERL, and ends in the residues valine-glutamate-aspartate-glutamate-cysteine (VEDEC). The Slo1QEERL form extends an additional ends in glutamine-glutamate-glutamate-argine-lysine (QEERL). These splice variants result in channels that have very similar gating properties.
The Slo1VEDEC forms tend to be retained in intracellular storage pools and only move to the cell surface upon stimulation of the cells with appropriate hormones or growth factors. By contrast, Slo1QEERL is expressed constitutively at high levels on the surface of cells even without treatment by hormones or growth factors. Slo1VEDEC can exert a dominant-negative function on the trafficking of heteromeric channels in which it is present.
Previous pharmacological strategies for manipulating the function of BKCa channels have been built around small molecules and natural-products/toxins that alter gating properties of channels or that affect the pore domains of the channel (primarily as inhibitors of ion flux). These effects are exerted on channels that are already localized at their normal positions in the cell membrane. There are limitations to this approach. Specifically, these molecules have tended to be non-specific in their actions, as they affect the gating of other types of channels, including calcium channels and chloride channels. Also, several have been reported to affect mitochondrial function and increase production of reactive oxygen species, and they are all lipophilic and therefore likely to cross the blood-brain barrier, resulting in a host of central effects that are likely to make the drugs poorly tolerated should they be used, for example, to treat erectile dysfunction.
Therefore, the purpose of the present invention is to overcome these limitations by designing, synthesizing, and testing small molecules that can affect the steady-state surface expression of BKCa channels by regulating trafficking and/or stability in the membrane. This is a different mode of action that can be made more specific than existing known mechanisms, and therefore leads to improved therapeutic outcomes.